Paths of Progress Spring/Summer 2012

How Scientists Solve Research Riddles

By Robert Levy

For every "a-ha!" moment in science, there are more than a few "hmmms."

The thrill of discovery is invariably preceded by periods of perplexity, when experiments yield confusing or contradictory results, a new approach becomes a dead end, or number-crunching leaves researchers numb.

Science is often portrayed as a dispassionate search for truth, which overlooks the passion that so many researchers bring to their work. What attracted many of them to the field, after all, is the opportunity to find new answers to knotty questions.

If the bench work of science rests on the methodical performance of well-defined procedures, the essence of scientific thinking is precisely that it is not routine, but often requires intuitive or logical leaps. The solution to a problem may lie in finding a new use for old technology, in viewing discrepancies from a different angle, or framing a question in a novel way.

At Dana-Farber, where cancer research and care are deeply entwined, the motivation for solving scientific puzzles is never purely intellectual, regardless of the modern focus on molecular networks and the genetic eccentricities of cells. The following stories help illustrate the stumbling blocks basic researchers often face, and the skills they use to overcome them.

For Brendan Price, PhD, inspiration came in the form of research from a previous generation of scientists.

Clues from the past

Nature's knack for packaging DNA in ultra-tight bundles within the cell nucleus poses a challenge for scientists like Dana-Farber's Brendan Price, PhD, who studies how cells repair breaks in their DNA.

Rather than jamming genetic material haphazardly into the nucleus, the cell neatly packs the molecular equivalent of a household's worth of clothing into a single suitcase. As part of this process, the cell wraps DNA around clusters of protein molecules called histones, forming a bead-like string of structures called nucleosomes.

The arrangement is so efficient that the DNA within it takes up about 250 times less space than if it were loose. Such compactness enables the cell to store six feet of DNA in a nucleus .0002 inches in diameter.

When a section of DNA breaks – as a result of exposure to radiation or chemicals, or through mistakes in cell division – cancer can result. In response, the cell dispatches several crews of proteins to fix it. The proteins thread their way through the nucleosomes, gently loosening them to clear a path to the broken section. Once there, they summon other proteins to piece the strands of DNA back together.

Ideally, researchers would mimic nature's own techniques for DNA repair. It isn't possible to replicate in a laboratory, however, a process that took thousands of years to evolve and involves dozens of different proteins – not all of which are known.

The question of how to locate the damaged regions where the nucleosomes have been unpacked absorbed scientists for years. At first, Price and his colleagues tried making artificial histones and tagging them with fluorescent tracers, hoping damaged cells would take in the fabricated histones and splice them into the damaged section of their DNA. All such attempts failed.

Then, inspiration struck. "I've always found it useful to go back and read the original studies to understand what investigators back then were doing," says Price. "It's a way of coming to the field fresh, without any preconceptions."

While perusing scientific articles from 30 and 40 years earlier, Price read that the strength of the DNA-histone bond varies with the level of salt in the surrounding fluid. By increasing the salt concentration, Price and his colleagues were able to weaken the DNA "wrapping" at the sites of damage, allowing them to remove the histones and DNA-repair proteins and giving them easy access to the damaged DNA. The technique proved so useful it has since been adopted by scientists around the world.

Kimberly Stegmaier, MD, found new uses for advanced technologies.

The undruggables

The inventor who realized that microwaves – originally used in radar and long-distance telephone communication – could be harnessed to cook food has something in common with Dana-Farber's Kimberly Stegmaier, MD.

Stegmaier and her colleagues have developed powerful tools for cancer drug discovery using techniques borrowed from other areas of biological research. Her area of interest lies deep within the cell's machinery for converting genetic information – stored in DNA – into proteins that carry out the cell's business.

Malfunctions in this machinery can give rise to a variety of cancers. Sometimes, researchers are able to identify the individual protein cogs at fault and block them with targeted drugs. In other cases, the guilty proteins are unknown, and even if they were known, they may be impossible to reach with current drugs – hence their reputation as "the undruggables."

Stegmaier devised a solution. "We asked ourselves, 'Are there technologies available now, which were not available 10 years ago, that could be used to screen large numbers of chemicals as potential cancer drugs?'" she remarks. "We realized that microarrays – DNA chips that genetically 'fingerprint' cells by charting the activity or inactivity of thousands of genes at a time – would fit the bill."

The Dana-Farber team used microarray technology to compare the genetic fingerprints of normal, mature cells with those of cancer cells. They then screened entire "libraries" of drugs to find which ones converted the cancer cells' fingerprint to normal.

At this point, however, another challenge loomed: While microarrays could be used to define genetic fingerprints, they weren't a practical way of screening many thousands of molecules at a reasonable price.

Again, the solution was the novel use of an existing technology – in this case, one that uses fluorescent beads to indirectly measure minute amounts of RNA, a courier of genetic information and an indicator of the activity level of specific genes. The technology meshed perfectly with the format used in drug screening.

Since then, Stegmaier's group has identified several compounds with promise for treating acute myeloid leukemia and other cancers. "Bringing these two technologies together was like pointing spotlights from two different angles on the problem," she remarks. "The intersection was the key to narrowing our search for drug candidates."

On the case in 3-D

Matthew Freedman, MD, and his colleagues had assembled a tidy case against a section of chromosome 24 as a co-conspirator in several kinds of cancer including colon, breast, and prostate.

The site, just a few units of DNA long, is located in what is known as a "gene desert," a region of chromosome devoid of genes that hold the code for making proteins. Without protein blueprints of its own, the site is thought to be involved in gene regulation – cranking the activity of nearby genes up or down to produce the proper amount of certain proteins.

Studies have shown that subtle variations at the site – changes in the placement of a handful of letters of the genetic code – are associated with many types of cancer. The question for scientists has been: Which gene or genes does the risk-increasing site regulate, and how?

Freedman and his associates thought they had the perfect candidate. The gene closest to the risk site is Myc (pronounced "mick"), one of the most notorious cancer-causing genes. The proximity suggested that the site interacts with Myc, and that changes in that interaction could have a role in cancer. Researchers needed only to show that variations in the risk site were linked to changes in Myc's activity, and the case would be solved.

However, Freedman's experiments found that the three major variations at the risk site produced no differences in Myc activity. "On paper and in theory, the connection seemed obvious, but we weren't able to prove it," Freedman relates. "We had to find another technique that would implicate Myc."

The alternative turned out to be chromosome conformational capture, a process that enables researchers to study how different parts of the genome interact in three dimensions. With this technique, Freedman discovered that the risk site has a "long-range loop" of DNA fiber that links to Myc like a molecular lasso.

"We demonstrated that these two regions are indeed co-localized [meaning they occupy the same space] and communicate with each other," Freedman says.

"Knowing that the risk site and Myc are in contact presents us with an attractive target for devising new cancer therapies."